The success of allogeneic (human to human) organ transplantation has been established in the last few decades, due to the limited supply of donor organs, many patients have little or no chance of receiving a transplanted organ, such as a kidney, heart or liver. A significant number of people die while awaiting an organ. Ungulate animals, such as porcine, ovine and bovine, are considered likely sources of xenograft organs and tissues. Porcine xenografts have been given the most attention since the supply of pigs is plentiful, breeding programs are well established, and their size and physiology are compatible with humans. However, there are several obstacles that must be overcome before the transfer of these organs or tissues into humans can be successful.
The immunological barriers to xenografts differ from those to allografts because of the greater molecular incompatibility between host and donor tissue. This results in a much greater role of the innate immune system, including naturally occurring antibodies, complement, and immune cells, in the rejection process than occurs in allotransplantation. This fundamental difference raises the height of the barrier considerably and is a major reason xenotransplantation is not a current clinical reality.
The first immunological hurdle is “hyperacute rejection” (HAR). HAR is defined by the ubiquitous presence of high titers of pre-formed natural antibodies binding to the foreign tissue. The binding of these natural antibodies to target epitopes on the donor tissue endothelium is believed to be the initiating event in HAR. This binding, within minutes of perfusion of the donor tissue with the recipient blood, is followed by complement activation, platelet and fibrin deposition, and ultimately by interstitial edema and hemorrhage in the donor organ, all of which cause rejection of the tissue in the recipient (Strahan et al. (1996) Frontiers in Bioscience 1, e34-41). The primary course of HAR in humans is the natural anti-galactose alpha 1,3-galactose antibody, which comprises approximately 1% of antibodies in humans and monkeys. Except for Old World monkeys, apes and humans, most mammals carry glycoproteins on their cell surfaces that contain the galactose alpha 1,3-galactose epitope (Galili et al., J. Biol. Chem. 263: 17755-17762, 1988). In contrast, glycoproteins that contain galactose alpha 1,3-galactose are found in large amounts on cells of other mammals, such as pigs. Humans, apes and old world monkeys do not express galactose alpha 1,3-galactose, but rather produce in high quantities a naturally occurring anti-galactose alpha 1,3-galactose antibody (Cooper et al., Lancet 342:682-683, 1993). It binds specifically to glycoproteins and glycolipids bearing galactose alpha-1,3 galactose. Alpha 1,3 galactosyltransferase is the enzyme that forms the galactose alpha-1,3 galactose epitopes on cells.
A recent, major breakthrough in the field of xenotransplantation was the production of the first live pigs lacking any functional expression of alpha 1,3 galactosyltransferase (Phelps et al. Science 299:411-414 (2003))
PCT publication No. WO 04/028243 by Revivicor, Inc. describes the successful production of viable pigs, as well as organs, cells and tissues derived therefrom, lacking any functional expression of alpha 1,3 galactosyltransferase. PCT Publication No. WO 04/016742 by Immerge Biotherapeutics, Inc. also describes the production of alpha 1,3 galactosyltransferase knock-out pigs.
The next significant barrier to xenotransplantation is delayed xenograft rejection, otherwise known as acute vascular rejection. This form of rejection invariably occurs in discordant vascularised xenografts in which HAR is prevented. The pathogenesis of delayed xenograft rejection, though poorly understood, is characterized by a distinct and often intractable inflammatory process, which can occur within 36-48 hours but typically occurs days to months after transplantation. Delayed xenograft rejection is characterized by the infiltration of recipient inflammatory cells and thrombosis of graft vessels, leading to ischaemia. Various strategies are currently under investigation to prevent delayed xenograft rejection, for example, PCT Publication No. WO 98/42850 by Imperial College discloses that the expression of coagulation inhibitors on the surface of the xenograft can inhibit the thrombotic aspect of this type of rejection.
The final major barrier encountered by xenografts is cell mediated rejection. The differences between recipients and allograft donor organs are largely restricted to small differences in the MHC antigens. There is predominantly direct recognition of these differences by host T cells and a predominantly Th1 type response occurs.
T-cell activation is involved in the pathogenesis of transplant rejection. Activation of T-cells requires at least two sets of signaling events. The first is initiated by the specific recognition through the T-cell receptor of an antigenic peptide combined with major histocampatibility complex (MHC) molecules on antigen presenting cells (APCs). The second set of signals is antigen nonspecific and is delivered by T-cell costimulatory receptors interacting with their ligands on APCs. In the absence of costimulation, T-cell activation is impaired or aborted, which may result in an antigen specific unresponsive state of clonal anergy, or in deletion by apoptotic death. Hence, the blockade of T-cell costimulation has been thought to provide an approach for suppressing unwanted immune responses in an antigen specific manner while preserving normal immune functions. (Dumont, F. J. 2004 Therapy 1, 289-304).
Of several T cell costimulatory pathways identified to date, the most prominent is the CD28 pathway. CD28, a cell surface molecule expressed on T-cells, and its counter receptors, the B7.1 (CD80) and B7.2 (CD86) molecules, present on dendritic cells, macrophages, and B-cells, have been characterized and identified as attractive targets for interrupting T-cell costimulatory signals. A second T-cell surface molecule homologous to CD28 is known as cytoxic T-lymphocyte associated protein 4 (CTLA4). CTLA4 is a cell surface signaling molecule, but contrary to the actions of CD28, CTLA4 negatively regulates T cell function. CTLA4 has 20-fold higher affinity for the B7 ligands than CD28. The gene for human CTLA4 was cloned in 1988 and chromosomally mapped in 1990 (Dariavach et al., Eur. J. Immunol. 18:1901-1905 (1988); Lafage-Pochitaloff et al., Immunogenetics 31:198-201 (1990); U.S. Pat. No. 5,977,318).
The CD28/B7 pathway has become an attractive target for interrupting T cell costimulatory signals. The design of a CD28/B7 inhibitor has exploited the endogenous negative regulator of this system, CTLA4. A CTLA4-immunoglobulin (CTLA4-Ig) fusion protein has been studied extensively as a means to inhibit T cell costimulation. A difficult balance must be reached with any immunosuppressive therapy; one must provide enough suppression to overcome the disease or rejection, but excessive immunosuppression will inhibit the entire immune system. The immunosuppressive activity of CTLA4-Ig has been demonstrated in preclinical studies of animal models of organ transplantation and autoimmune disease.
Soluble CTLA4 has recently been tested in human patients with kidney failure, psoriasis and rheumatoid arthritis. Bristol-Myers Squibb's drug Abatacept, soluble CTLA4-Ig has recently been approved for the treatment of rheumatoid arthritis. This drug is the first in the new class of selective T cell costimulation modulators. Bristol-Myers Squibb is also conducting Phase II clinical trials with Belatacept (LEA29Y) for allograft kidney transplants. LEA29Y is a mutated form of CTLA4, which has been engineered to have a higher affinity for the B7 receptors than wild-type CTLA4, fused to immunoglobulin. Repligen Corporation is also conducting clinical trials with its CTLA4-Ig for idiopathic thrombocytopenic purpura.
Although CTLA-4 from one organism is able to bind to B7 from another organism, the highest avidity is found for allogeneic B7. Thus, while soluble CTLA-4 from the donor organism can thus bind to both recipient B7 (on normal cells) and donor B7 (on xenotransplanted cells), it preferentially binds B7 on the xenograft. Thus, for applications in xenotransplantation, particularly pig to human, porcine CTLA4 could be used to induce immunosuppression. PCT Publication No. WO 99/57266 by Imperial College discloses the porcine CTLA4 sequence and the administration of soluble CTLA4-Ig for xenotransplantation therapy. Vaughn A. et al., Journal of Immunology (2000) 3175-3181, describes binding and function assays demonstrating species specificity in the action of soluble porcine CTLA4-Ig.
To date, much of the research on CTLA4-Ig as an immunosuppressive agent has focused on administering soluble forms of CTLA4-Ig to a patient. Transgenic mice engineered to express CTLA4-Ig have been created and subject to several lines of experimentation. Ronchese et al. examined immune system function generally after expression of CTLA4 in mice (Ronchese et al. J Exp Med (1994) 179: 809; Lane et al. J Exp Med. 1994 Mar. 1; 179(3):819). Sutherland et al. (Transplantation. 2000 69(9):1806-12) described the protective effect of CTLA4-Ig secreted by transgenic fetal pancreas allografts in mice to test the effects of transgenically expressed CTLA4-Ig on allogenic islet transplantation. Lui et al. (J Immunol Methods 2003 277: 171-183) reported the production of transgenic mice that expressed CTLA4-Ig under control of a mammary specific promoter to induce expression of soluble CTLA4-Ig in the milk of transgenic animals for use as a bioreactor.
PCT Publication No. WO 01/30966 by Alexion Pharmaceuticals, Inc. describes chimeric DNA constructs containing the T cell inhibitor CTLA-4 attached to the complement protein CD59, as well as transgenic porcine cells, tissues, and organs containing the same.
Martin C. et al., Transgenic Research (2005) 14: 373-384, describes transgenic fetal porcine neurons that express human CTLA4-Ig under the control of the neuron-specific enolase promoter for use in the cellular transplantation of neurons to treat human neurodegenerative disorders.
It is object of the present invention to provide ungulate organs, cells and tissues which decrease the immune response of humans on transplantation.
It is another object of the present invention to provide methods to decrease the immune response of humans on transplantation of ungulate organs, cells and tissues.